FIG. 2.

Construction and confirmation of recombinant BACs. (A) Recombinant BACs were constructed by homologous recombination of a NheI-linearized DNA fragment with Towne BAC, kindly provided by F. Lui, University of California, Berkeley. UL121 and UL128 served as flanking regions to introduce targeted mutations into exon 5 of IE2. The UL127 locus was replaced by the CAT reporter, as described previously (27, 30). A kanamycin resistance (Kan^r) cassette was inserted between UL127 and UL128 for selection of recombinant BACs. (B) The integrity of recombinant BACs was verified by digesting BAC DNA with the HindIII restriction enzyme. (C) Exon 5 of IE2 was amplified from the recombinant BACs by PCR and digested with the indicated restriction enzyme. Successful recombination of the Q548A or Q548R mutation introduced a new KasI or EagI restriction site, respectively, compared to WT.